Molecular markers of malva sylvestris and their application

Abstract

This invention provides SSR molecular markers for Hibiscus rosa-sinus and their applications, belonging to the field of molecular marker technology. The invention comprises 15 molecular markers, with corresponding primer sequences shown in SEQ ID NO. 1~30. Testing revealed that this series of markers exhibits a 100% polymorphism rate, excellent polymorphism information content, marker index, and resolution, and strong genetic stability. The molecular marker primers of this invention can efficiently distinguish Hibiscus rosa-sinus germplasm materials, clearly revealing the population's genetic structure and phylogenetic relationships. They are suitable for Hibiscus rosa-sinus germplasm resource identification, genetic diversity analysis, molecular fingerprinting construction, and assisted breeding, possessing the characteristics of high specificity, high resolution, and wide applicability.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, and in particular to the Hibiscus SSR molecular marker and its applications. Background Technology

[0002] Hibiscus rosa-sinensis, also known as Chinese hibiscus, is an evergreen shrub or small tree belonging to the genus Hibiscus in the Malvaceae family. It is widely distributed in tropical and subtropical regions. Hibiscus flowers boast a rich and vibrant array of colors, including red, pink, yellow, white, and purple. Flower types include single, double, and semi-double petals. With a long flowering period, blooming continuously throughout the year, it has high application value in landscaping, potted plants, and cut flower production, making it a preferred tree species for urban greening and garden beautification. Meanwhile, hibiscus has significant ecological value. Its dense leaves and strong transpiration can effectively regulate the local climate and purify the air. It also has a certain resistance and adsorption capacity to harmful gases such as sulfur dioxide. In the field of medicine, the flowers, leaves, and roots of hibiscus can all be used as medicine, with effects such as clearing heat and dampness, cooling blood and detoxifying, reducing swelling and relieving pain. It is often used to treat diseases such as lung heat cough, dysentery, carbuncles and boils. In addition, the petals of hibiscus are rich in natural pigments and can be used as food additives or cosmetic raw materials. The nectar can also be used to brew beverages, showing potential development prospects in the light industry sector.

[0003] Although Hibiscus rosa-sinensis has important ornamental, ecological, medicinal, and economic value, research on its genetic resources is relatively lagging. Although there are some molecular marker studies, the existing molecular markers have low polymorphism information content and limited genomic loci coverage, which cannot meet the needs of analyzing the complex genetic diversity of Hibiscus rosa-sinensis. At the same time, the repeatability and stability of the markers are poor and they are easily affected by factors such as PCR reaction conditions and primer concentration, resulting in insufficient accuracy in identifying genetic differences between varieties. Furthermore, in recent years, the continuous expansion of the market demand for Hibiscus rosa-sinensis has led to the widespread development of artificial hybridization breeding. However, due to the lack of effective genetic marker tools, the definition of kinship among varieties is unclear, and the location of genes related to superior traits is difficult, resulting in a breeding cycle of 3 to 5 years and low breeding efficiency. At the same time, some Hibiscus rosa-sinensis varieties have the same name but different species, and the same species has different names, which makes the protection of variety rights lack precise identification basis. In addition, the habitat of wild Hibiscus rosa-sinensis germplasm resources has been destroyed in the process of urbanization, and genetic diversity is at risk of loss. Therefore, it is urgent to develop more SSR molecular markers with high specificity and high polymorphism to conduct a more comprehensive evaluation of the genetic diversity of Hibiscus rosa-sinensis genetic resources and provide technical support for variety identification, molecular-assisted breeding, and germplasm resource protection. Summary of the Invention

[0004] In view of this, the present invention provides an SSR molecular marker primer set for identifying Hibiscus varieties or detecting the genetic diversity of Hibiscus germplasm resources and its application. Using the SSR molecular marker primer set described in this invention, different Hibiscus varieties can be accurately screened and identified, effectively promoting the breeding process of Hibiscus.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides SSR molecular markers for Hibiscus rosa-sinus, which are numbered as follows: 643-P2, 643-P15, 643-P16, 643-P23, 643-P24, 643-P27, 643-P41, 643-P42, 643-P57, 643-P59, 643-P61, 643-P62, 643-P88, 643-P93 and 643-P97. The primer sequences corresponding to each molecular marker are shown in SEQ ID NO.1~30.

[0007] The present invention also provides specific primers for amplifying the molecular markers, including sequences as shown in SEQ ID NO.1~30.

[0008] The present invention also provides a kit comprising the aforementioned specific primers.

[0009] The present invention also provides a hibiscus genome chip containing the aforementioned molecular markers.

[0010] This invention also provides the application of the primers or the kit described herein in the construction of molecular fingerprints of Hibiscus rosa-sinensis.

[0011] This invention also provides the application of the primers or kits described herein in the genetic diversity analysis of Hibiscus germplasm resources.

[0012] The present invention also provides the application of the primers or kits described herein in the identification of Hibiscus varieties, analysis of kinship, analysis of population genetic structure, or tracing of maternal lineage.

[0013] This invention also provides the application of the primers or the kits described herein in molecular marker-assisted breeding of Hibiscus rosa-sinensis.

[0014] Preferably, the application includes the following steps:

[0015] (1) Extract DNA from the Hibiscus rosa-sinensis sample to be tested;

[0016] (2) Using the DNA extracted in step (1) as a template, SSR PCR amplification was performed using the specific primers described above;

[0017] (3) SSR PCR products were detected using a capillary electrophoresis system.

[0018] Preferably, the SSR PCR amplification reaction system is as follows: ddH2O 14.8 μL, dNTP 0.4 μL, Buffer 2 μL, upstream primer 0.3 μL, downstream primer 0.3 μL, DNA template 2 μL, Taq enzyme 0.2 μL; each reaction system contains one pair of primers;

[0019] The reaction program for SSR PCR amplification was as follows: pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 54℃ for 35 s, extension at 72℃ for 40 s, for a total of 35 cycles; and final extension at 72℃ for 3 min.

[0020] By adopting the above technical solution, the present invention has the following beneficial effects: The present invention uses 15 molecular markers, numbered 643-P2, 643-P15, 643-P16, 643-P23, 643-P24, 643-P27, 643-P41, 643-P42, 643-P57, 643-P59, 643-P61, 643-P62, 643-P88, 643-P93, and 643-P97, with corresponding primer sequences shown in SEQ ID NO. 1~30. Testing revealed that this series of markers exhibits a 100% polymorphism rate, excellent polymorphism information content, marker index, and resolution, and strong genetic stability. The molecular marker primers of this invention can efficiently distinguish Hibiscus germplasm materials, clearly reveal the population genetic structure and kinship, and are suitable for Hibiscus germplasm resource identification, genetic diversity analysis, molecular fingerprinting construction and assisted breeding. They have the characteristics of high specificity, high resolution and wide applicability. Attached Figure Description

[0021] Figure 1 This is an agarose gel electrophoresis image of a portion of the 100 primer pairs amplified in 10 hibiscus varieties.

[0022] Figure 2 PAGE gel electrophoresis images of some of the 50 selected primer pairs amplified in 10 hibiscus varieties.

[0023] Figure 3 This is a fluorescence capillary electrophoresis genotyping peak diagram of the amplification products of some Hibiscus rosa-sinensis samples using primer P15.

[0024] Figure 4 This is a fluorescence capillary electrophoresis genotyping peak diagram of the amplification products of some Hibiscus rosa-sinensis samples using primer P57.

[0025] Figure 5 This is a UPGMA clustering dendrite of 140 hibiscus germplasms constructed based on the amplification results of 15 pairs of SSR primers.

[0026] Figure 6 This is a population genetic structure analysis diagram of 140 hibiscus germplasms based on 15 pairs of SSR markers at K=5. Note: Different colors in the diagram represent different genetic components (K=5), each vertical bar represents one hibiscus germplasm, and the proportion of different colors in the vertical bar corresponds to its genetic contribution rate from each genetic component. Detailed Implementation

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1. Development of SSR molecular markers for Hibiscus rosa-sinensis

[0029] The hibiscus transcriptome data were obtained from NovaSeq X Plus high-throughput sequencing of flowers, leaves, and young stems of two hibiscus varieties. The materials used for sequencing came from two hibiscus varieties, as detailed in Table 1.

[0030] Table 1. Information on the source of materials for Hibiscus rosa-sinensis transcriptome sequencing

[0031] Variety Name Geographical location latitude and longitude altitude Number of individuals Hibiscus with split petals No. 8 Anyang Road, Xixiangtang District, Nanning City, Guangxi 108°18′E 22°52′N 74-79 1 Green pole hanging bell No. 8 Anyang Road, Xixiangtang District, Nanning City, Guangxi 108°18′E 22°52′N 74-79 1

[0032] Note: 1 represents "independent plant individual", and the number of samples taken from each plant individual is 3.

[0033] Flowers, leaves, and young stems of the two hibiscus varieties were selected and mixed in equal amounts for transcriptome sequencing. Based on the SSR loci identified in the transcriptome sequencing, MISA (http: / / pgrc.ipkgatersleben.de / misa / ) software was used for SSR locus screening and analysis. The screening criteria were set as follows: repeat unit length 1–6 bp; the minimum repeat counts for single nucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide units were 10, 6, 5, 5, 5, and 5, respectively. Based on these screening criteria, batch primer design was conducted, and 100 primer pairs (numbered P1–P100) were randomly selected to perform PCR experiments on 10 hibiscus varieties (Table 2). PCR products were screened for primer amplifiability using 1.5% agarose gel electrophoresis. The amplification results of some primers are shown below. Figure 1 As shown, 50 pairs of primers with clear bands were selected from 100 pairs for 6% denaturing polyacrylamide gel electrophoresis to screen for polymorphic primers (if all sample bands are in basically the same position, it indicates poor primer polymorphism; if all sample bands are in different positions, it indicates good primer polymorphism). The amplification results of some primers are shown below. Figure 2As shown in Table 2 (samples were spotted from left to right according to the numbers in Table 2), 15 pairs of primers with clear bands and good polymorphism were selected, as shown in Table 3.

[0034] Table 2 Information on 10 Hibiscus varieties used for PCR primer screening

[0035] Serial Number Variety Name Geographical location latitude and longitude Altitude (m) Number of individuals 1 Hibiscus with split petals No. 8 Anyang Road, Xixiangtang District, Nanning City, Guangxi 108°18′E 22°52′N 74-79 1 2 Green pole hanging bell No. 8 Anyang Road, Xixiangtang District, Nanning City, Guangxi 108°18′E 22°52′N 74-79 1 3 Arnault No. 8 Anyang Road, Xixiangtang District, Nanning City, Guangxi 108°18′E 22°52′N 74-79 1 4 Colorful Hibiscus Hibiscus germplasm resource nursery next to Nanning Shitong Cement Products Co., Ltd., Shibu Town, Xixiangtang District, Nanning City, Guangxi 108°11′E 22°48′N 74-79 1 5 Graf Medusa Hibiscus germplasm resource nursery next to Nanning Shitong Cement Products Co., Ltd., Shibu Town, Xixiangtang District, Nanning City, Guangxi 108°11′E 22°48′N 74-79 1 6 203-3 Hibiscus germplasm resource nursery next to Nanning Shitong Cement Products Co., Ltd., Shibu Town, Xixiangtang District, Nanning City, Guangxi 108°11′E 22°48′N 74-79 1 7 54-4 Hibiscus germplasm resource nursery next to Nanning Shitong Cement Products Co., Ltd., Shibu Town, Xixiangtang District, Nanning City, Guangxi 108°11′E 22°48′N 74-79 1 8 Rose Nebula Hibiscus germplasm resource nursery next to Nanning Shitong Cement Products Co., Ltd., Shibu Town, Xixiangtang District, Nanning City, Guangxi 108°11′E 22°48′N 74-79 1 9 C199-23 Hibiscus germplasm resource nursery next to Nanning Shitong Cement Products Co., Ltd., Shibu Town, Xixiangtang District, Nanning City, Guangxi 108°11′E 22°48′N 74-79 1 10 Hongta Hibiscus germplasm resource nursery next to Nanning Shitong Cement Products Co., Ltd., Shibu Town, Xixiangtang District, Nanning City, Guangxi 108°11′E 22°48′N 74-79 1

[0036] Note: 1 represents "independent plant individual", and the number of samples taken from each plant individual is 3.

[0037] Table 3 SSR primer sequences

[0038]

[0039] Example 2. Validation of Hibiscus SSR molecular markers in a population

[0040] The 67 hibiscus accessions include: *Hibiscus rosa-sinensis*, ... Medusa's Eye, Red Velvet, White Mist and Red Dust, Queen of Spades, Sweet Taro Cake, Rouge Tears, Burning Heart of the Caribbean, Dream City, Apollina, Upper River, Orange Flying, Malaysian Apricot, Cinderella, Black Mochi, Beauty Under the Moon, Coolberry, Imakuretis, Red Dragon Hibiscus, Vermilion Red, Fire Phoenix, Drunken Red Butterfly, White Cloud Turmeric, Columbine, Dreaming of Tahiti, White Jade Butterfly, Double Yellow, Single Red, Single Orange Red, Orange Love Song, Red Dream, Daihong Starry Sky Reverie.

[0041] After synthesizing fluorescent primers for 15 pairs of highly polymorphic primers, PCR experiments were performed on the above 67 hibiscus germplasms and 73 hibiscus hybrid offspring (Table 4). After the PCR products passed the agarose gel electrophoresis test, they were detected by capillary electrophoresis. Formamide and molecular weight internal standard ROX-500 were mixed at a volume ratio of 0.5 μL: 8.5 μL, and the PCR product volume was 1 μL. Then, capillary electrophoresis was performed using a 3730XL sequencer.

[0042] Table 4. Parental information of 73 hibiscus hybrid offspring

[0043] serial number Variety Name Father Mother 9 203-3 Purple Peony Flower Language of Beauty 10 54-4 Rising Sun Diamond Lemon 12 C199-3 Purple Peony Lemon Black Tea ZJ-13 C90-8 Orange Love Song White Jade Butterfly 14 C11-9 Shiva 2711 15 FC7-3 73-2 60-6 17 C365-2 Purple Lotus Shiva 18 C26-7 Heart of the Ocean White Jade Butterfly 25 C135-2 201902-13 White Jade Butterfly 29 C216-7 1706 Rouge Tears 30 FC31-13 2-1 60-6 31 FC16-6 112-3 158-4 32 C62-3 Cherry Blossom Queen unknown 33 C209-17 Purple Peony Lemon Black Tea 34 C289-12 Shiva 2711 35 C324-4 13# 9# 36 C15-2 201902-13 20# 37 C206-2 Heart of the Ocean White Jade Butterfly 38 C483-3 Dreaming of Tahitian 1712 39 C307-4 Shiva Flower Language of Beauty 40 C199-5 Purple Peony Lemon Black Tea 42 C327-1 Purple Lotus Jasmine Hanging Gardens 43 C432-13 Dreamy Neon Dress Chocolate cake 44 C182-4 Cherry Blossom Queen Fairy Zixia 45 D349-10 10-2 Rouge Tears 46 D171-3 153-1 B17-6 47 D294-9 Pacific Ocean 2-1 48 D246-11 Blue neon lights 13-4 52 B162-8 Flower Language of Beauty Orange Love Song 62 FC16-2 112-3 158-4 63 C153-1 14# White Jade Butterfly 66 As one wishes Dream City Orange Love Song 72 C457-5 181225 201902-02 73 Nian Lan Shuang Shiva Fairy Zixia 74 C173-10 Heart of the Ocean White Jade Butterfly 75 C98-2 Rouge Tears Fairy Zixia 76 C124-2 The Burning Heart of the Caribbean Fairy Zixia 77 C32-3 Shiva Cherry Blossom Queen 78 C400-15 Purple Lotus Shiva 79 FC13-12 73-2 60-6 80 C143-8 Orange Love Song Fairy Zixia 81 C270-2 The Burning Heart of the Caribbean Lemon Black Tea 82 C349-3 White Jade Butterfly Jasmine Hanging Gardens 83 C207-2 Yellow butterfly 1705 84 C60-4 Cherry Blossom Queen Fairy Zixia 85 C262-1 Dream City Fairy Zixia 86 C262-2 Dream City Fairy Zixia 99 230-2 Flower Language of Beauty Lemon Black Tea 100 200-1 Purple Peony Flower Language of Beauty 101 D294-5 Pacific Ocean 2-1 102 D53-1 Song of Autumn 1705 103 D59-6 B223-8 181225 104 D302-1 Rouge Tears 88-9 115 D63-5 Mysterious Lake 31-5 116 D190-9 Purple Lotus Night Elf 117 D332-2 1913 Rouge Tears 121 FC27-3 84-12 24-14 122 D381-4 94-2 Shiva 123 C75-34 Shiva 181225 124 C23-6 Dreaming of Tahitian Rainbow under the moonlight 125 C448-8 White Jade Butterfly Chocolate cake 126 B161-1 Flower Language of Beauty Orange Love Song 127 C488-4 13# Shiva 131 2-1 Rising Sun Su Mei 132 C488-6 White Jade Butterfly Chocolate cake 133 C6-4 Purple Lotus 8# 134 C432-11 Dreamy Neon Dress Chocolate cake 135 C400-2 Purple Lotus Shiva 136 C148-4 Cherry Blossom Queen Fairy Zixia 137 C477-9 Chocolate cake Snowy Love 138 C253-2 Moonlight Romance 1712 139 C406-10 14# Yonghong 140 C153-3 14# White Jade Butterfly

[0044] The SSR PCR reaction system consisted of 20 μL of the following components: 14.8 μL ddH2O, 0.4 μL dNTPs, 2 μL Buffer, 0.3 μL (20 μM) upstream primer, 0.3 μL (20 μM) downstream primer, 2 μL DNA template, and 0.2 μL Taq enzyme.

[0045] SSR PCR amplification program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 35 s, 72℃ extension for 40 s, for a total of 35 cycles; final extension at 72℃ for 3 min.

[0046] Capillary electrophoresis: Using a GeneScan 500LIZ as an internal reference, the PCR products were subjected to capillary electrophoresis on a 3730XL DNA analyzer to separate the amplified fragments. The fluorescence capillary electrophoresis genotyping peaks of the SSR PCR amplification products from some samples using primers 643-P15 and 643-P57 are shown in the figure. Figure 3 and Figure 4 As shown.

[0047] The raw data obtained from the sequencer were analyzed using the Fragment analysis function in Genemarker V2.2.0 software. The positions of the molecular weight internal standards in each lane were compared with the positions of the peak values ​​of each sample to obtain the fragment size.

[0048] Using a primer pair as one allele locus, the fragment size (i.e., phenotype) of each sample at each allele locus was entered into an Excel file according to the format required by Convert 1.31 software. Then, Convert 1.31 software was used to convert the data to the format required by POPGENE software. Genetic diversity analysis of each primer and population was performed using POPGENE32 software. The results of the genetic diversity analysis are shown in Table 5, and the comprehensive evaluation statistics of molecular marker polymorphism are shown in Table 6.

[0049] Table 5 Results of genetic diversity analysis

[0050] Locus Statistical types Effective sample size Effective number of alleles (Ne) Nei's genetic diversity index (H) Shannon Information Index (I) Polymorphic sites Polymorphism percentage (%) P2 average value 140 1.4484 0.2611 0.393 7 100 P2 Standard deviation 0.4004 0.2066 0.2834 P15 average value 140 1.3796 0.2266 0.358 5 100 P15 Standard deviation 0.4165 0.2029 0.2598 P16 average value 140 1.4937 0.2734 0.4077 6 100 P16 Standard deviation 0.46 0.2248 0.2965 P23 average value 140 1.3407 0.2109 0.3311 5 100 P23 Standard deviation 0.3723 0.1978 0.2737 P24 average value 140 1.4422 0.2516 0.3783 10 100 P24 Standard deviation 0.4214 0.2116 0.286 P27 average value 140 1.2353 0.146 0.239 7 100 P27 Standard deviation 0.3575 0.1825 0.2526 P41 average value 140 1.2143 0.1271 0.2017 10 100 P41 Standard deviation 0.3561 0.1893 0.2642 P42 average value 140 1.1372 0.0832 0.1344 11 100 P42 Standard deviation 0.2856 0.1643 0.2333 P57 average value 140 1.4541 0.2555 0.3856 8 100 P57 Standard deviation 0.4439 0.2153 0.2849 P59 average value 140 1.4747 0.283 0.4278 13 100 P59 Standard deviation 0.3506 0.1808 0.2471 P61 average value 140 1.0945 0.0712 0.1338 9 100 P61 Standard deviation 0.1697 0.1106 0.1678 P62 average value 140 1.1486 0.0998 0.1684 8 100 P62 Standard deviation 0.2497 0.156 0.2275 P88 average value 140 1.4103 0.2366 0.3621 7 100 P88 Standard deviation 0.429 0.2117 0.2825 P93 average value 140 1.2398 0.1362 0.21 7 100 P93 Standard deviation 0.3958 0.2104 0.2905 P97 average value 140 1.2614 0.1637 0.2647 9 100 P97 Standard deviation 0.3467 0.1811 0.2522

[0051] Genetic diversity analysis of 15 molecular marker loci (Table 5) showed that the percentage of polymorphic loci in the 140 tested materials reached 100%, indicating excellent marker polymorphism. The mean values ​​of the effective allele count, Shannon information index, and Nei's genetic diversity index were generally low, indicating that the overall genetic diversity level of the experimental materials was moderate to low, and the genetic basis was relatively narrow. Significant differences in genetic diversity were observed among different loci. Loci P57, P2, and P24 showed abundant variation, while loci P59, P61, and P62 exhibited lower genetic diversity, indicating significant differentiation among loci.

[0052] Table 6. Statistical Table of Polymorphism Comprehensive Evaluation

[0053] Locus polymorphism level Polymorphic Information Content (PIC) Effective number of polymorphic sites (EMR) Marked Index MI Resolution RP P2 0.754723 7 5.283063 2.814286 P15 0.62447 5 3.122349 1.885714 P16 0.717133 6 4.302799 2.414286 P23 0.635003 5 3.175019 2.014286 P24 0.82536 10 8.253604 4.215827 P27 0.615067 7 4.306457 1.057143 P41 0.662506 6 6.25063 2.214286 P42 0.522431 11 5.746738 1.228571 P57 0.777437 8 6.219498 2.9 P59 0.880864 13 11.45123 6.171429 P61 0.406988 9 3.66289 0.7 P62 0.477865 8 3.822917 1.028571 P88 0.735643 7 5.149502 2.585714 P93 0.536162 7 3.753131 0.657143 P97 0.735166 9 6.616498 2.685714

[0054] A comprehensive analysis of the polymorphism of 15 molecular markers (Table 6) showed that 13 of the tested markers had high polymorphism levels (PIC > 0.5) and excellent mean polymorphic information content (PIC). Only two loci showed moderate polymorphism. Significant differences were found in marker index (MI) and resolution (RP). P59 and P24 showed the best polymorphism, effective band count, and overall identification ability, making them the core preferred markers. P61 and P93 loci showed slightly weaker overall performance. Overall, the markers can be effectively used for subsequent genetic diversity analysis and variety identification studies of germplasm resources.

[0055] The genetic identity (I) and genetic distance (D) of Nei (1972) were used to measure the degree of genetic differentiation among populations. Based on the genetic distance coefficient, a UPGMA tree was constructed using MEGA 11, and after secondary refinement on the website https: / / itol.embl.de / , a clustering diagram was generated, as shown below. Figure 5 As shown.

[0056] Figure 5 This is a circular clustering tree constructed based on molecular marker data. Different colored branches in the figure represent different genetic groups. Figure 5 The results showed that 15 pairs of SSR primers could classify 140 tested Hibiscus rosa-sinensis materials into multiple independent groups based on genetic relationships. The cluster structure was clear, and the group boundaries were distinct, indicating significant genetic differentiation among the materials. Materials within the same group showed high genetic similarity and close kinship; materials between different groups had greater genetic distance and obvious population structure characteristics. The clustering results were consistent with previous analyses of genetic diversity and polymorphism, validating the effectiveness of the tested molecular markers and the reliability of the experimental data. The overall genetic background of the materials exhibited characteristics of intra-group conservation and inter-group differentiation. This demonstrates that the 15 pairs of SSR primers can effectively distinguish all tested germplasm, providing a reliable molecular marker reference for Hibiscus rosa-sinensis germplasm identification, genetic diversity analysis, and molecular-assisted breeding.

[0057] Using STRUCTURE 2.3.4 and an Admixture model, the initial number of non-counting iterations for MCMC (Markov's chain Monte Carlo) was set to 50,000, followed by another 50,000 iterations. K was set from 2 to 10, with each K iteration run 10 times. Finally, a suitable K value was selected based on ΔK, calculated as the difference in LnP(D) between adjacent K values. The Structure results were further processed using the Structure Harvester online tool and CLUMMP software, and finally, the results were graphically displayed using Distruct software. The genetic structure diagram of the Structure run with K=5 is shown below. Figure 6 As shown.

[0058] Figure 6 The results showed that the 140 *Hibiscus rosa-sinensis* accessions could be divided into several subpopulations with significantly different genetic backgrounds. Each subpopulation exhibited a distinct, compartmentalized distribution, and the population structure was clear and stable. The vast majority of individuals in the tested population showed a single genetic component, indicating a pure genetic background and high genetic isolation between subpopulations. Only a very small number of individuals showed inter-subpopulation gene infiltration, resulting in a low overall level of gene flow. These results are highly consistent with the grouping patterns of phylogenetic clustering trees, further confirming the significant genetic differentiation of the tested materials. This demonstrates that 15 pairs of SSR primers can effectively resolve the population genetic structure of the tested *Hibiscus rosa-sinensis* germplasm, providing important molecular genetic evidence for *Hibiscus rosa-sinensis* germplasm identification, genetic diversity evaluation, and molecular breeding.

[0059] As can be seen from the above embodiments, the present invention provides Hibiscus SSR molecular markers and their applications. The molecular markers of the present invention have the characteristics of high specificity, high resolution and wide applicability.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Hibiscus SSR molecular marker, characterized in that, The molecular markers are numbered as follows: 643-P2, 643-P15, 643-P16, 643-P23, 643-P24, 643-P27, 643-P41, 643-P42, 643-P57, 643-P59, 643-P61, 643-P62, 643-P88, 643-P93 and 643-P97. The specific primer sequences corresponding to each molecular marker are shown in SEQ ID NO.1~30.

2. A specific primer for amplifying the molecular marker of claim 1, characterized in that, This includes sequences such as those shown in SEQ ID NO.1~30.

3. A reagent kit, characterized in that, Includes the specific primers described in claim 1.

4. A hibiscus genome chip, characterized in that, Contains the molecular marker as described in claim 1.

5. The application of the Hibiscus SSR molecular marker combination of claim 1, the specific primer of claim 2, the kit of claim 3, or the Hibiscus genome chip of claim 4 in the construction of Hibiscus molecular fingerprinting.

6. The application of the primers of claim 2 or the kit of claim 3 in the genetic diversity analysis of Hibiscus germplasm resources.

7. The application of the primers of claim 2 or the kit of claim 3 in the identification of Hibiscus varieties, analysis of kinship, analysis of population genetic structure or maternal pedigree tracing.

8. The application of the primers of claim 2 or the kit of claim 3 in molecular marker-assisted breeding of Hibiscus rosa-sinensis.

9. The application according to any one of claims 5 to 8, characterized in that, Includes the following steps: (1) Extract DNA from the Hibiscus rosa-sinensis sample to be tested; (2) Using the DNA extracted in step (1) as a template, SSR PCR amplification was performed using the specific primers described in claim 2; (3) SSR PCR products were detected using a capillary electrophoresis system.

10. The application according to claim 9, characterized in that, The SSR PCR amplification reaction system consisted of: 14.8 μL ddH2O, 0.4 μL dNTPs, 2 μL Buffer, 0.3 μL upstream primer, 0.3 μL downstream primer, 2 μL DNA template, and 0.2 μL Taq enzyme; each reaction system contained one pair of primers. The SSR PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 54℃ annealing for 35 s, 72℃ extension for 40 s, for a total of 35 cycles; and finally 72℃ extension for 3 min.

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